The kinetics of carbon monoxide (CO) binding to cytochromes P450 was measured using laser flash-photolysis techniques. An apparatus was constructed to photolyze CO from P450 and to monitor the subsequent recombination reaction. Signal-processing techniques, in conjunction with a newly developed difference kinetic analytical method, were applied to define the kinetic behavior of individual P450s. P450s in liver microsomes from rats treated with various drugs and carcinogens were examined. These agents exerted a wide range of effects on their target P450s. In addition, single human P450s expressed by means of baculovirus were examined. Kinetic analysis revealed that these single P450s are composed of multiple species with differing sensitivities to drugs and carcinogens.